Dokument: Development of a modular platform for the tailor-made biosynthesis of lignans: From monolignols towards (-)-podophyllotoxin
| Titel: | Development of a modular platform for the tailor-made biosynthesis of lignans: From monolignols towards (-)-podophyllotoxin | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=57875 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20211125-081542-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Decembrino, Davide [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Urlacher, Vlada [Gutachter] Jun.-Prof. Dr. Axmann, Ilka M. [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Plants secondary metabolic pathways are source of a broad plethora of bioactive compounds that have been widely employed in both modern and traditional medicine. In this regard, lignans are prominent examples, as they have long been attributed to several health promoting effects on the human body. Traditional means of production involve the extraction of lignans from Forsythia, Linum, Sesamum and Podophyllum plant species, however the increasing commercial demand determined overexploitation and environmental endangerment of the sources. Total-synthesis routes have been developed as alternatives; however, their feasibility is hampered by the high complexity and stereochemistry of the compounds. In search for more sustainable approaches, intensive genome mining led to the disclosure of many enzymes composing the lignans secondary metabolic network in planta. Complementary, advances in metabolic engineering and synthetic biology allowed the assembly of such pathways in recombinant bacteria and yeast.
In this thesis, E. coli was used as host to harbour the biosynthetic route starting from coniferyl alcohol and proceeding towards the microtubules depolymerizer (-)-podophyllotoxin. In total, ten enzymes were assembled to develop combinable, independent modular units targeting the biosynthesis of diverse lignans such as (±)-pinoresinol, (-)-matairesinol, (-)-pluviatolide, and (-)-deoxypodophyllotoxin at need. Given the fundamental role within plant secondary metabolism, special attention was given to the recombinant expression of plant cytochrome P450 monooxygenases because reconstituting their activity in prokaryotic host is not trivial. Within this work, three P450 enzymes originating from Sinopodophyllum hexandrum were successfully expressed in active state in E. coli for the first time. In the first developmental step within this work, the biotransformation of the monolignol coniferyl alcohol to the lignan (-)-matairesinol was achieved. E. coli’s endogenous multi-copper oxidase CueO was employed after copper-mediated activation to furnish (±)-pinoresinol via coniferyl alcohol dimerization. The kinetic resolution of (±)-pinoresinol represents the first branching point in lignan biosynthesis and it was here performed by the IX pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR). The secoisolariciresinol dehydrogenase from Podophyllum peltatum (PpSDH) was implemented as a further step to produce (-)-matairesinol. In the second step, the biotransformation of (+)-pinoresinol to the non-commercially available lignan (-)-pluviatolide was achieved at a preparative scale. To produce (-)-pluviatolide, (-)-matairesinol is oxidized by the action of a methylenedioxy bridge-forming P450 from Sinopodophyllum hexandrum (CYP719A23) with high regio- and enantioselectivity. To assemble a 4-step multi-enzyme cascade, FiPLR and PpSDH were coexpressed with CYP719A23, whose activity was supported and optimized by employing the NADPH-dependent reductase ATR2 from Arabidopsis thaliana. After the optimization of this setup, the efficient conversion of (+)-pinoresinol was achieved leading to the isolation of 137 mg/L product with 76% isolated yield and high purity (>99% UV/vis, >94% MS, ee ≥99%). In the third step, the previous multi-enzyme cascade was prolonged by assembling an effective 5-steps biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin (98% yield), further extendable to (-)-epipodophyllotoxin. In this setup, among the seven enzymes involved, activity of three plant P450s, CYP719A23, CYP71CU1, and CYP82D61 could be reconstituted in E. coli. To date, a physiological putative (-)-podophyllotoxin synthase from S. hexandrum performing the last step – the hydroxylation of (-)-deoxypodophyllotoxin to (-)-podophyllotoxin -remains unidentified. Alternatively, two potential candidates from the P450 library of ourgroup (CYP107Z and CYP105D from Streptomyces platensis) were evaluated for their ability to hydroxylate (-)-deoxypodophyllotoxin. Both P450s were found active, and (-)-podophyllotoxin and (-)-epipodophyllotoxin were detected as reaction products. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie | |||||||
| Dokument erstellt am: | 25.11.2021 | |||||||
| Dateien geändert am: | 25.11.2021 | |||||||
| Promotionsantrag am: | 15.06.2021 | |||||||
| Datum der Promotion: | 27.09.2021 |
